Programming of Development

Question 1

When does development begin?

Answer 1

-development commences with the production of a zygote following fertilisation

Question 2

Understanding Development in Humans

Problems

Answer 2

• humans are very complex
• ordered progression of development is dependent on the expression of the genetic information encoded in the genome
• how is this information expressed at the right time, in the right places to lead to the formation of a normally functioning organism

Question 3

Understanding Development in Humans

Solution

Answer 3

• don’t start with studying humans as they are too complex

- identify simple model organisms amenable to experiment and relate their information to more complex organisms

Question 4

Animal Development

Model Organisms

Answer 4

• fruit fly - Drosophilia melanogaster
• nematode worm - C. elegans
• mouse - Mus musculus
• zebra fish - Danio rerio
• cress - Arabidopsis thaliana

Question 5

Questions in Understanding Development

Answer 5

• what are the mechanisms that control development?
• how is an organism programmed to develop shape and form characteristic of its species?
• how do the individual parts of organisms develop to perform different functions
• genetic analysis and molecular biology combined could provide answers

Question 6

Cellular Division and Differentiation

Answer 6

• a parent cell divides to form two daughter cells
• in development, the 2 daughter cells are different in function because they express different sets of genes in the genome
• but the different genes expressed are mostly consequences of differentiation, not the cause
• we can identify the genes that regulate developmental decisions by isolating mutants in which the process is disrupted i.e. a parent cell that divides to produce to identical daughter cells

Question 7

Bacteriophage Development

Answer 7

• a simple prokaryotic model
• bacteriophage development requires programming of gene expression in time
• in many bacteriophages, expression of some genes occurs early in the maturation process, in other genes it is late
• this is achieved by a cascade mechanism, expression of early genes is required to allow the expression of late genes
• mechanisms controlling the switch between early and late gene expression vary in different viruses

Question 8

Examples of Early-Late Gene Switches

Answer 8

• in phage λ , the product of an early gene, Q, is a transcription factor required to allow the transcription of later genes
• in phage T7, gene 1 encodes an RNA polymerase which can only transcribe from late gene promoters, RNA polymerase produced by the host (E.coli) is used to transcribe the early genes
• in phage T4, early genes 33 and 55 produce transcription factors needed for the transcription of later genes

Question 9

Bacteriophage Morphogenesis

Answer 9

• assembly of phage T4 capsid is an excellent example of how mutants can establish the order of events in a morphogenesis pathway
• mutants can be isolated that are blocked in capsid assembly
• these need to be conditional mutants, no capsid = no virus

Question 10

Morphology of Bacteriophage T4

Answer 10

• capsid assembly can be divided into three major steps, head, tail and tail fibres
• the tail is composed of the base plate, core and sheath
• the head is attached to the top of the tail
• the tail fibres are connected to the base plate

Question 11

Bacteriophage T4 Assembly

Stages

Answer 11

1) the empty head is assembled, DNA is inserted, more proteins are added to complete the head
2) base plate is synthesised, core forms, sheath forms and is built up around the core
3) tail fibres are assembled in stages from precursor molecules
- steps 1, 2, and 3 take place concurrently, there is evidence for the order of assembly and independence of the three steps from mutant analysis
- the head is attached to the tail, and the tail fibres are attached to the base plate

Question 12

Bacteriophage T4 Assembly

Extra Information

Answer 12

• not all gene products are structural components
• some of the non-structural proteins are assembly enzymes
• the non-structural proteins are less abundant
• historically they were called minor proteins as less of them is made but this is misleading as their role in assembly is NOT minor

Question 13

Bacteriophage T4 Assembly

Head

Answer 13

• 24 gene products are required to make a head
• 10 are structural
• gene 23 encodes a major head capsid protein
• mutants blocked in head formation are still able to assemble tails and tail fibres
• some capsid associated proteins are nucleases responsible for cutting up DNA into ‘headful lengths’ as DNA is synthesised as a concatemer

Question 14

Bacteriophage T4 Assembly

Analysis Approach - Description

Answer 14

• identification of temperature sensitive mutants blocked in the different stages (head, tail and fibre) of capsid assembly
• use complementation analysis (double infections) to determine how many genes control each stage
• use biochemical analysis (SDS-polyacrylamide gel electrophoresis) to identify proteins defective in each mutant

Question 15

Bacteriophage T4 Assembly

Analysis Approach - Head Example

Answer 15

• gene 22 is required to assemble the empty head
• if gene 22 is blocked, a headless phage is produced
• under the microscope, clumps or protein 22 are visible as well as assembled tail and fibre complexes

Question 16

Bacteriophage T4 Assembly

Base Plate and Tail Assembly

Answer 16

• assembly of the base plate and tail involves similar principles to head assembly
• 32 genes are required, 26 of which are structural
• mutants blocked in tail sheath production can make heads but heads cant be joined to the defective tail
• similarly mutants can also make tail fibres but cant join them to defective tails

Question 17

Bacteriophage T4 Assembly

Analysis Approach - Tail Example

Answer 17

• P18 is the principle sheath protein, required for initiation of sheath formation
• when P18 is blocked, under the microscope you can see core-base plate complexes and complete heads which aren’t attached to the tail
• the tail is visibly thinner than usual as there is no sheath

Question 18

Bacteriophage T4 Assembly

Tail Fibre Assembly

Answer 18

• mutants blocked in tail fibre production can make heads and tails and can join these together
• but viable phage particles as tail fibres cant be formed and attached

Question 19

Bacteriophage T4 Assembly

Analysis Approach - Tail Fibre Example

Answer 19

• gene 57 encodes a tail fibre pre cursor
• there are no subtail fibres, so no tail fibres
• under a microscope, head-tail complexes with naked base plates can be seen

Question 20

Programming of Development

Summary

Answer 20

• many different genes may be differentially expressed as a result of developmental change
• but only a few genes cause developmental transitions
• isolation of mutants in which developmental transitions are blocked is a powerful means of identifying these causative genes
• a combination of genetic and biochemical approaches are necessary to identify how developmental transitions are implemented

Question 21

Development in Complex Eukaryotes - Synopsis

Answer 21

• Drosophilia melanogaster is an outstanding model for animal developmental studies
• homoeotic mutations define key regulatory genes
• C. elegans provides an alterative strategy

Question 22

Drosophilia

Life Cycle

Answer 22

• egg
• larva (maggot)
• pupa (formation of organs)
• adult fly

Question 23

Drosophilia

Adult Body

Answer 23

• segmented
• divided into three major sections, head, thorax an abdomen
• has a head & tail and a top & bottom (front&back)
• these assymetries are developed very early in development
• bilaterally symmetrical

Question 24

Drosophilia

Genes That Determine Symmetry - Using Mutants

Answer 24

• mutants are isolated in which establishment of the axes of symmetry has failed
• some of these mutation are recessive lethal, but can be studied up until the point of death
• mutation stocks can be maintained as heterozygotes, these give 1/4 mutant progeny upon intercrossing
• but some mutations are ‘maternal effect’ in nature

Question 25

Drosophilia | Maternal Effect Genes

Answer 25

- the earliest stages of development are programmed by genes that are active in the mother cells, maternal effect genes - they mutate to effect embryo development, i.e. if the mother is homozygous for the mutation then all of her offspring will be abnormal - the fathers (sperm) genotype is irrelevant at this stage - the embryos genotype is also unimportant in its own development, the embryo could be homozygous for the mutation but as long as the mother isn't it will develop normally

Question 26

Drosophilia | Establishment of Embryonic Polarity

Answer 26

- very early event in embryo development - two axes are established, anterior posterior (head to tail) and dorsal ventral (front to back) - maternal effect mutations lead to embryos with defective polarity

Question 27

Drosophilia | Early Embryogenesis

Answer 27

- repeated nuclear mitosis without cellularisation - the early embryo is coenocytic, the multiple nuceli share one cytoplasm - the oocyte is surrounded by maternal nurse cells - the nurse cells deliver the products of the maternal genes (mRNA and proteins) into the developing embryo

Question 28

Drosophilia | Snake - Mutant

Answer 28

- a maternal effect mutation leading to abnormal dorsal-ventral polarity - at least 10 maternal effect genes have been identified which can mutate to affect the dorsal-ventral axis of embryonic symmetry - mutation of the snake gene leads to very abnormal embryos with dorsal structures all around the body

Question 29

Drosophilia | Snake - Wildtype Gene Product

Answer 29

- the wildtype snake gene product is required for correct establishment of the dorsal-ventral axis of symmetry - the mutant dorsal phenotype caused by snake can be repaired by injecting cytoplasm from a wildtype embryo into young mutant embryos - this repair also occurs if wildtype snake mRNA is injected - snake mutants repaired by injection can form larvae that go on to become fertile flies - the snake gene is not expressed from the embryonic genome, it is the maternal genotype that matters as mRNA is supplied to the early embryo by the nurse cells

Question 30

Drosophilia | Dorso-Ventral Asymmetry - Toll

Answer 30

- established in response to a dorsal-ventral morphogenetic gradient - one of the dorsal genes Toll shows different responses to micro injection - for other dorsal mutations injection position of wildtype cytoplasm doesn't matter and a normal dorsa-ventral axis is established - for Toll mutants the new ventral surface is always established at the site of the injection - Toll encodes a transmembrane protein receptor of spätzle and allows import of other proteins into the embryo - Snake and other proteases release spätzle from a protein complex inside the embryo - spätzle binds with Toll so Toll can import the dorsal transcription factor into the nuclei - Toll is only present in the membrane on the dorsal side so when the dorsal transcription factor enters the cell, it diffuses through the cell creating a concentration gradient

Question 31

Drosophilia | Anterior Posterior Axis - Overview

Answer 31

- regulated by the bicoid (bcd) gene - a maternal effect gene - offspring from bcd/bcd homozygous mothers have neither head nor thorax

Question 32

Drosophilia | Anterior-Posterior Axis - Bicoid

Answer 32

- the bicoid gene is transcribed in maternal cells at the anterior end of the egg - bcd mRNA passes into the oocyte through cytoplasmic canals where it is trapped by linkage to cytoskeleton - after fertilisation, bcd mRNA is translated and the produced protein diffuses from the anterior pole forming an anterior to posterior gradient but is always at its highest concentration at the anterior end

Question 33

Drosophilia | Anterior-Posterior Axis - Nanos

Answer 33

- (nos) - second maternal mRNA - deposited at the posterior end - its protein product forms posterior to anterior gradient in the fertilised egg - it blocks expression of the genes that bcd activates - Nos mutants fail to form abdominal structures

Question 34

Drosophilia | Development and Embryonic Genes

Answer 34

- a fundamental part of Drosophilia development (establishment of polarity) is not determined by its own genes - embryonic genes are however responsible for transducing the mothers asymmetry and conveying it to the embryo - i.e. the segments in the embryo have to be 'aware' of where on the axis they lie - the maternal role is not central in all systems but is important in many - in other species, environmental inputs e.g. gravity, light, sperm penetration ... are required for orderly development

Question 35

Drosophilia | Antennapedia

Answer 35

- a homoeotic mutation - legs in the place of antennae - genes specifying the development of secondary legs are expressed in place of antennae - positional information is misinterpreted

Question 36

Drosophilia | Ultrabithorax

Answer 36

- ubx - one of a group of genes regulating segmentation - ubx mutants produce structures on the 2nd segment of the thorax that should be on the 1st - instead of halteres, wings are formed - duplication of the 1st segment (and no '2nd' segment)

Question 37

Drosophilia | Homeobox Genes

Answer 37

- genes which mutate to produce homoeotic phenotypes - e.g. antennapedia and ubx - all located in a gene cluster on the chromosome - encode proteins with a highly conserved amino acid domain, the DNA binding domain of the protein - this domain, the hox domain, is highly conserved in evolution

Question 38

Hox Genes and Conservation in Animal Evolution

Answer 38

- Drosophilia homoeotic genes are expressed in the region of the body that they control - segmentation genes encode DNA-binding transcription factors that contain a common sequence motif, the hox domain - mammals have similar genes that are also expressed postitionally - mouse homologues of ubx are expressed in a similar median position along the developing embryo as in flies - this suggests that programming along the anterior-posterior axis evolved very early in animal evolution

Question 39

C. elegans

Answer 39

- a nematode worm - 1st animal to have its genome sequenced - small, ~1000 cells - has cell types typical of more complex animals - easily cultured - eats E.coli - transparent, so all cells can be viewed microscopically - 2 sexes, male (XO) and hermaphrodite (XX) - hermaphrodites can be self fertilised or crossed with males - easily transformed allowing cell specific gene expression patterns to be identified by delivering GFP/RFP... fusions

Question 40

C. elegans Development

Answer 40

- unlike in drosophilia where cell fate is determined by receiving morphogenetic signals from cellular environment, most developmental processes in C.elegns are pre-programmed - the fate of the cell is dependent on its ancestry rather than position relative to other genes - the fertilised egg divides into an anterior and posterior cell - these cells divide again and the 4 cell embryo is composed of 2 anterior and 2 posterior cells - the 2 posterior cells specifically express the PAL-1 gene, a homeobox transcription factor that regulates development of posterior structures